5-methylcytosine (m5C) occurs in the context of CpG dinucleotides and is the most abundant covalently modified base in the genomes of vertebrates. Areas of high CpG dinucleotide density, so called ‘CpG islands’, are spread throughout the genomes and usually map to gene promoter regions. Methylation of promoter CpG islands is associated with histone deacetylation and transcriptional silencing (1), and is essential for normal embryonic development, genomic imprinting and X-chromosome inactivation. Recently, somatic de novo methylation of CpG islands in tumor suppressor genes has been implicated in tumorigenesis, and aberrant methylation of imprinted genes is associated with several inherited human diseases (1-3). The central role of DNA methylation in normal and disease-related processes has resulted in a variety of methods to detect and characterise normal and aberrant methylation patterns in biological and clinical specimens.
In standard PCR and cloning procedures, information about m5C and other covalent base modifications in genomic DNA is lost. Therefore, current PCR methods for detecting and mapping m5C in specific genes rely on treatment of genomic DNA with methylation-sensitive restriction endonucleases or sodium bisulfite prior to amplification. Bisulfite converts unmethylated cytosines to uracil, while methylated cytosines remain unreactive (4). A specific target sequence can subsequently be amplified with primers specific for bisulfite-converted DNA and examined for its m5C content. The golden standard among bisulfite methods is genomic sequencing that provides a positive display of m5C at specific CpG sites in virtually any stretch of DNA (5). More simple methods using bisulfite-converted DNA as template include methylation-specific PCR (MSP) (6), methylation-sensitive single nucleotide primer extension (7) and procedures based on the use of restriction endonucleases (8;9).
Despite the obvious advantages of the above methods, they all entail a two-step procedure, comprising initial PCR amplification and subsequent product analysis, usually by gel electrophoresis. Furthermore, with the exception of genomic sequencing, they are limited to the analysis of one or a few CpG sites in each setting.